How Automating Agarose Gel Fragment Selection and Sample Analysis Works

How Automating Agarose Gel Fragment Selection and Sample Analysis Works

Problem: Molecular biology relies on the ability to precisely target and amplify nucleic acids, and next-generation sequencing (NGS) platforms and cloning reactions benefit from precise size selection and analytical characterization of samples. For decades, researchers have used electrophoresis with agarose gels for both size selection and fragment-length distribution assessment of DNA samples for downstream assays.

However, in the new era of big science and high-throughput sample processing, the manual work required for electrophoresis is becoming harder to sustain. The associated errors have forced labs with high-throughput pipelines to use new approaches.

Today’s modest labs are conducting at least 3,000 sequencing reactions per year for translational medicine studies, and rerun rates spurred by manual errors can cost millions of dollars.

As a result, upfront sample quality control tests that rely on rapid characterization are crucial for many applications, including miRNA-seq, RNA-seq, ChIP-seq, long-fragment sequencing, gene synthesis and cloning. Reliable size selection for these samples is also critical in maximizing the value of downstream processes.

The manual process for gel-based size selection can cause sample-to-sample result deviation. The processes of gel casting, target identification, and gel-dissolution recovery of nucleic acids are all subject to variability. Furthermore, manual size selection is an involved process that can add a full day or more to genomics workflows.

Solution: Hamilton Robotics’ recently launched automated solution on Microlab® NIMBUS® Select workstation using Coastal Genomics' Ranger Technology is one example of how the process is being improved.

Coastal Genomics' Ranger Technology updates both the agarose gel size selection and analytics processes to meet the needs of genomics laboratories. Combined with the Hamilton Robotics workstation, this automated solution removes operator variability, reduces sample preparation time by collapsing the analytical processes of the assay into the size selection process and secures sample integrity for downstream processes.

The NIMBUS Select workstation is a small-footprint liquid handler that can size-select as few as one or as many as 96 samples in a single run. In addition, inexpensive, high-resolution analytics cassettes can be used to characterize up to 192 samples in a run without size selection. The NIMBUS has validated protocols for the downstream library prep for most major NGS manufacturers.

Unlike traditional agarose gel band selection, Ranger Technology does not need UV light to illuminate the bands. Throughout the electrophoretic process, DNA bands are illuminated by exposure to visible light. Ranger Technology images these bands to identify and recover target fractions, thereby avoiding DNA damage from UV exposure and preventing any potential impact on downstream applications.

The workstation can size-select 96 samples in two hours, shaving six hours off of other gel extraction protocols. Users define the base pair range of their target fragment and the workstation automatically loads the samples from a 96-well source plate into the Ranger agarose gel cassettes. The gantry-mounted camera records multiple images of the electrophoretic process. These images are analyzed to identify the target, which is recovered by the workstation when it arrives at an extraction well in the agarose gel cassette. Electropherograms before and after the size-selection event are reported for all samples.

Ranger Technology offers improvements over bead-based approaches. Additional QC analytical processes required for beads are consolidated into the size-selection run. The system typically improves recovery yields and is capable of size selection on targets exceeding 20 kbp. Users can expect to eliminate undesirable off-target fragments, increase read lengths and improve normalization of libraries prior to sequencing. Ranger Technology's analytics capability is ideal for groups faced with the requirements to assess fragment length distributions. Up to 192 high-resolution electropherograms can be developed in less than one hour. The system flexibly accommodates runs in which only a fraction of the capacity are loaded, thereby saving consumable costs. Coastal Genomics is also developing a low-resolution analytics solution that will accommodate up to 384 analyses per run.

This new and affordable method for size selection and analytics leverages automation to eliminate labor-intensive protocols. As the workload for gene synthesis and NGS increases, this system promises to save researchers valuable time and money.

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